Activation of Peptide Prodrugs by hK2

ABSTRACT

The invention provides novel peptide prodrugs that contain cleavage sites specifically cleaved by human kallikrein 2 (hK2). These prodrugs are useful for substantially inhibiting the non-specific toxicity of a variety of therapeutic drugs. Upon cleavage of the prodrug by hK2, the therapeutic drugs are activated and exert their toxicity. Methods for treating cell proliferative disorders are also featured in the invention.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 11/725,135, filed on Mar. 15, 2007 (abandoned), which is acontinuation application of U.S. application Ser. No. 11/212,028, filedon Aug. 24, 2005 (abandoned), which is a continuation application ofU.S. application Ser. No. 09/627,600, filed on Jul. 28, 2000 (now U.S.Pat. No. 7,053,042), which claims the benefit of U.S. ProvisionalApplication No. 60/146,316, filed Jul. 29, 1999, each of which is herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention relates generally to the targeted activation ofbiologically active materials to cells that produce human glandularkallikrein (hK2) and more specifically to hK2-specific peptidessubstrates that can act as drug carriers. In addition it relates toprodrugs consisting of a peptide covalently coupled to a cytotoxic drugsuch that the peptide-drug bond can be hydrolyzed by hK2. The couplingof the peptide to the cytotoxic drug creates an inactive prodrug thatcan only become activated at sites where enzymatically active hK2 isbeing produced.

BACKGROUND OF THE INVENTION

There is currently no effective therapy for men with metastatic prostatecancer who relapse after androgen ablation, even though numerous agentshave been tested over the past thirty years. Prolonged administration ofeffective concentrations of standard chemotherapeutic agents is usuallynot possible because of dose-limiting systemic toxicities.

Human Glandular Kallikrein 2 (hK2) is the protein product of the humankallikrein gene hKLK2, one of three related kallikrein genes that alsoinclude hKLK1 and hKLK3. These three genes are clustered on chromosome19q13.2-q13.4. The protein product of hKLK3 is prostate-specific antigen(PSA). While PSA is the predominant tissue kallikrein in the prostate,hK2 is also found almost exclusively in the prostate. hK2 is aglycoprotein containing 237 amino acids and a mass of 28.5 kDa. hK2 andPSA share some properties such as high amino acid sequence identity,prostate localization, androgen regulation and gene expression, but arequite distinct form one another biochemically.

hK2 and PSA differ most markedly in their enzyme properties. Unlike PSA,a chymotrypsin-like protease, hK2 displays the trypsin-like specificitycommon to most members of the kallikrein family of proteases. hK2 cancleave semenogelin proteins, with an activity that is comparable to PSA.The level of hK2 in the seminal fluid is only 1% of the level of PSA.hK2 has trypsin-like activity, similar to hK1, although it does notappear to function as a classic kininogenase.

In the normal prostate, the levels of expressed hK2 protein are lowerthan those of PSA. However, hK2 is more highly expressed by prostatecancer cells than by normal prostate epithelium. Comparison ofimmunohistochemical staining patterns demonstrated incrementallyincreased staining in poorly differentiated prostate cancers. Theintensity of staining has been found to increase with increasing Gleasonscore, in contrast to PSA, which tends to show decreased staining withincreasing Gleason grade, suggesting that hK2 might potentially be abetter tumor marker for prostate cancer than PSA.

Recently, three independent groups reported that recombinant hK2 couldconvert inactive pro-PSA in to the mature PSA protease by release of thepropeptide in vitro, thus establishing a possible physiologic connectionbetween hK2 and PSA. hK2 is also secreted in an inactive precursor form.Pro-hK2 may have autocatalytic activity, but the mechanism of activationin vivo is unknown and may involve several additional enzymes. hK2 hasalso been shown to activate single chain urokinase-type plasminogenactivator, scuPA, to the active two-chain form, uPA, which is highlycorrelated with prostate cancer metastasis. More recently, hK2 has beenshown to inactivate the major tissue inhibitor of uPA, plasminogenactivator inhibitor-1 (PAI-1). Thus hK2 may influence the progression ofprostate cancer by the activation of uPA and by the inactivation ofPAI-1.

Enzymatically active hK2 has also been shown to form covalent complexesin vitro with plasma protease inhibitors such as α₁-antichymotrypsin(ACT), α₂-antiplasmin, antithrombin III, protein C inhibitor (PCI), andα₂-macroglobulin (AMG). hK2 has been identified in prostate cancer serumin a complex with ACT.

Thapsigargin (TG) is a sesquiterpene-y-lactone available by extractionfrom the seeds and roots of the umbelliferous plant Thapsia garganica L.Thapsigargin selectively inhibits the sarcoplasmic reticulum (SR) andendoplasmic reticulum (ER) Ca²⁺-ATPase (SERCA) pump, found in skeletal,cardiac, muscle and brain microsomes. The apparent dissociation constantfor TG from the SERCA pump is 2.2 pM or less.

SUMMARY OF THE INVENTION

The present invention provides a novel class of oligopeptides thatinclude amino acid sequences containing cleavage sites for humanglandular kallikrein (hK2) (see FIG. 1). These cleavage sites arederived from an hK2 specific cleavage map of semenogelin I and II, (seeFIG. 1). These oligo-peptides are useful in assays that can determinethe free hK2 protease activity. Furthermore, the invention also providesa therapeutic prodrug composition, comprising a therapeutic drug linkedto a peptide, which is specifically cleaved by hK2. The linkagesubstantially inhibits the non-specific toxicity of the drug, andcleavage of the peptide releases the drug, activating it or restoringits non-specific toxicity.

The invention also provides a method for treating cell proliferativedisorders, including those which involve the production of hK2, insubjects having or at risk of having such disorders. The method involvesadministering to the subject a therapeutically effective amount of thecomposition of the invention.

The invention also provides a method of producing the prodrugcomposition of the invention. In another embodiment, the inventionprovides a method of detecting hK2 activity in tissue. In yet anotherembodiment, the invention provides a method of selecting appropriateprodrugs for use in treating cell proliferative disorders involvinghK2-production.

The invention also provides a method for detecting a cell proliferativedisorder associated with hK2 production in a tissue of a subject,comprising contacting a target cellular component suspected of having ahK2 associated disorder, with a reagent which detects enzymaticallyactive hK2.

The invention also provides a method of determining hK2 activity in ahK2-containing sample, comprising contacting the sample with adetectably labeled peptide which is specifically cleaved by hK2 for aperiod of time sufficient to allow hK2 to cleave the peptide, detectingthe detectable label to yield a detection level, which is then comparedto the detection level obtained by contacting the same detectablylabeled peptide with a standard hK2 sample of known activity.

The invention also provides a method of imaging soft tissue and/or bonemetastases which produce hK2, comprising administering alipophilic-imaging label linked to a peptide which is specificallycleaved by hK2 to a patient suspected of having a hK2-associated cellproliferative disorder, allowing hK2 to cleave the peptide, allowing thelipophilic imaging label to accumulate in the tissue and/or bone,allowing the subject to clear the uncleaved peptide, and imaging thesubject for diagnostic purposes.

Unless otherwise defined, all technical and scientific terms used hereinhave the ordinary meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other reference materials mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a portion of the amino acid sequence of Semenogelin I (SEQ IDNOs: 1-4) and Semenogelin II (SEQ ID NOs: 5-11), showing the cleavagesites for human kallikrein 2.

DETAILED DESCRIPTION

The invention provides a novel class of peptides that contain a cleavagesite specific for human glandular kallikrein 2 (hK2). These peptides areefficiently and specifically cleaved by hK2. These peptides are usefulfor substantially inhibiting the non-specific toxicity of thetherapeutic agents prior to the agents contracting a tissue containinghK2. Thus, the invention includes prodrugs which include peptides linkedto therapeutic agents. The prodrugs of the invention comprise peptidesequences containing a cleavage site specific for hK2 and therapeuticdrugs. The compositions do not show significant non-specific toxicity,but in environments where hK2 is found, the composition becomesactivated when peptide is cleaved, releasing the therapeutic drug, whichregains its non-specific toxicity.

hK2 Specific Peptide

As used herein the term “human glandular kallikrein 2” (hK2) means humanglandular kallikrein 2, as well as other proteases that have the same orsubstantially the same proteolytic cleavage specificity as hK2. As usedherein, the term “naturally occurring amino acid side chain” refers tothe side chains of amino acids known in the art as occurring inproteins, including those produced by post translational modificationsof amino acid side chains. The term “contacting” refers to exposingtissue to the peptides, therapeutic drugs or prodrugs of the inventionso that they can effectively inhibit cellular processes, or kill cells.Contacting may be in vitro, for example by adding the peptide, drug orprodrug to a tissue culture to test for susceptibility of the tissue tothe peptide, drug or prodrug. Contacting may be in vivo, for exampleadministering the peptide, drug, or prodrug to a subject with a cellproliferative disorder, such as prostate or breast cancer. By“polypeptide” is meant any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). As written herein, amino acid sequences are presentedaccording to the standard convention, namely that the amino-terminus ofthe peptide is on the left, and the carboxy terminus on the right. Inone aspect the invention features a peptide containing an amino acidsequence that includes a cleavage site specific for hk2 or an enzymehaving a proteolytic activity of hK2. The peptides of the invention arepreferably not more than 20 amino acids in length, more preferably tomore than ten amino acids in length. The preferred amino acid sequencesof the invention are linear. In an embodiment of the invention the aminoacid sequence may be cyclical such that the cyclical form of thesequence is an inactive drug that can become an activated drug uponcleavage by hK2 and linearization.

The cleavage site recognized by hK2 is flanked by at least an amino acidsequence, X₄X₃X₂X₁. This oligopeptide contains the amino acid arginine,histidine or lysine at position X₁. X₂ can be arginine, phenylalanine,lysine, or histidine. X₃ can be lysine, serine, alanine, histidine orglutamine. X₄ can be from 0 to 20 further amino acids, preferably atleast two further amino acids. Some preferred embodiments include asequence for X₄ that is substantially identical to the 20 amino acids inthe wild type semenogelin I or semenogelin II sequence that are the fromfourth to twenty fourth amino acids to the N-terminal side of recognizedsemenogelin cleavage sites. The amino acid sequence can further compriseX⁻¹, which is linked to the carboxy terminus of X₁ to create the aminoacid sequence X₄X₃X₂X₁X⁻¹. X⁻¹ is up to a further 10 amino acids, andcan include any amino acids. Preferably X₁ has leucine, alanine orserine linked to the carboxy terminus of X₁. X⁻¹ can include L- orD-amino acids. The hK2 cleavage site is located at the carboxy terminalside of X₁.

In some preferred peptides, both X₁ and X₂ are arginine.

Some examples of preferred peptides include (note that the symbol ][denotes an hK2 cleavage site):

(SEQ ID NO: 12)  1. Lys-Arg-Arg][ (SEQ ID NO: 13)  2. Ser-Arg-Arg][(SEQ ID NO: 14)  3. Ala-Arg-Arg][ (SEQ ID NO: 15)  4. His-Arg-Arg][(SEQ ID NO: 16)  5. Gln-Arg-Arg][ (SEQ ID NO: 17)  6. Ala-Phe-Arg][(SEQ ID NO: 18)  7. Ala-Gln-Arg][ (SEQ ID NO: 19)  8. Ala-Lys-Arg][(SEQ ID NO: 20)  9. Ala-Arg-Lys][ (SEQ ID NO: 21) 10. Ala-His-Arg][

Additional preferred peptides of longer sequence length include:

(SEQ ID NO: 22) 11. Gln-Lys-Arg-Arg][ (SEQ ID NO: 23)12. Lys-Ser-Arg-Arg][ (SEQ ID NO: 24) 13. Ala-Lys-Arg-Arg][(SEQ ID NO: 25) 14. Lys-Lys-Arg-Arg][ (SEQ ID NO: 26)15. His-Lys-Arg-Arg][ (SEQ ID NO: 27) 16. Lys-Ala-Phe-Arg][(SEQ ID NO: 28) 17. Lys-Ala-Gln-Arg][ (SEQ ID NO: 29)18. Lys-Ala-Lys-Arg][ (SEQ ID NO: 30) 19. Lys-Ala-Arg-Lys][(SEQ ID NO: 31) 20. Lys-Ala-His-Arg][

Additional preferred peptides that include an X⁻¹ amino acid are:

(SEQ ID NO: 32) 21. Lys-Arg-Arg][Leu (SEQ ID NO: 33)22. Ser-Arg-Arg][Leu (SEQ ID NO: 34) 23. Ala-Arg-Arg][Leu(SEQ ID NO: 35) 24. Ala-Arg-Arg][Ser (SEQ ID NO: 36)25. His-Arg-Arg][Ala (SEQ ID NO: 37) 26. Gln-Arg-Arg][Leu(SEQ ID NO: 38) 27. Ala-Phe-Arg][Leu (SEQ ID NO: 39)28. Ala-Gln-Arg][Leu (SEQ ID NO: 40) 29. Ala-Lys-Arg][Leu(SEQ ID NO: 41) 30. Ala-Arg-Lys][Leu (SEQ ID NO: 42)31. Ala-His-Arg][Leu

Preferred peptides of still longer sequence length having X⁻¹ include:

(SEQ ID NO: 43) 32. His-Ala-Gln-Lys-Arg-Arg][Leu (SEQ ID NO: 44)33. Gly-Gly-Lys-Ser-Arg-Arg][Leu (SEQ ID NO: 45)34. His-Glu-Gln-Lys-Arg-Arg][Leu (SEQ ID NO: 46)35. His-Glu-Ala-Lys-Arg-Arg][Leu (SEQ ID NO: 47)36. Gly-Gly-Gln-Lys-Arg-Arg][Leu (SEQ ID NO: 48)37. His-Glu-Gln-Lys-Arg-Arg][Ala (SEQ ID NO: 49)38. Gly-Gly-Ala-Lys-Arg-Arg][Leu (SEQ ID NO: 50)39. His-Glu-Gln-Lys-Arg-Arg][Ser (SEQ ID NO: 51)40. Gly-Gly-Lys-Lys-Arg-Arg][Leu (SEQ ID NO: 52)41. Gly-Gly-His-Lys-Arg-Arg][Leu

Other embodiments of peptide sequences which are useful for cleavage byhK2 and proteases with the hydrolytic activity of hK2 are disclosed inthe Examples section. Further examples of the peptides of the inventionare constructed as analogs of, derivatives of and conservativevariations on the amino acids sequences disclosed herein. Thus, thebroader group of peptides having hydrophilic and hydrophobicsubstitutions, and conservative variations are encompassed by theinvention. Those of skill in the art can make similar substitutions toachieve peptides with greater activity and or specificity toward hK2.For example, the invention includes peptide sequences described above,as well as analogs or derivatives thereof, as long as the bioactivity ofthe peptide remains. Minor modifications of the primary amino acidsequence of the peptides of the invention may result in peptides thathave substantially equivalent activity as compared to the specificpeptides described herein. Such modifications may be deliberate, as bysite directed mutagenesis or chemical synthesis, or may be spontaneous.All of the peptides produced by these modifications are included herein,as long as the biological activity of the original peptide remains, i.e.susceptibility to cleavage by hK2.

Further, deletion of one or more amino acids can also result in amodification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule without significantly alteringits biological activity. This can lead to the development of a smalleractive molecule which would also have utility. For example, amino orcarboxy-terminal amino acids which may not be required for biologicalactivity of the particular peptide can be removed. Peptides of theinvention include any analog, homolog, mutant or isomer or derivative ofthe peptides disclosed in the present invention, as long as bioactivitydescribed herein remains. All peptides described have sequencescomprised of L-amino acids; however, D-forms of the amino acids can besynthetically produced and used in the peptides described herein.

The peptides of the invention include peptides which are conservativevariations of those peptides specifically exemplified herein. The term“conservative variation” as used herein denotes the replacement of anamino acid residue by another, biologically similar residue. Examples ofconserved variations include the substitution of one hydrophobic residuesuch as isoleucine, valine, leucine, alanine, cysteine, glycine,phenylalanine, proline, tryptophan, tyrosine, norleucine or methioninefor another or the substitution of one polar residue for another such asthe substitution of arginine for lysine or histidine, glutamic foraspartic acids or glutamine for asparagine, and the like. Neutralhydrophilic amino acids that can be substituted for one another includeasparagine, glutamine, serine, and threonine. Such conservativesubstitutions are within the definitions of the classes of peptides ofthe invention with respect to X positions which may be any number ofamino acids. The peptides that are produced by such conservativevariation can be screened for suitability of use in the prodrugs of theinvention according to the methods for selecting prodrugs providedherein.

A wide variety of groups can be linked to the carboxy terminus of X₁ orX⁻¹. Notably, therapeutic drugs can be linked to this position. In thisway advantage is taken of the hK2 specificity of the cleavage site, aswell as other functional characteristics of the peptides of theinvention. Preferably, the therapeutic drugs are linked to the carboxyterminus of X₁ either directly or through a linker group. The directlinkage is preferably through an amide bond, in order to utilize theproteolytic activity and specificity of hK2. If the connection betweenthe therapeutic drug and the amino acid sequence is made through alinker, this connection is also preferably made through an amide bond,for the same reason. This linker may be connected to the therapeuticdrug through any of the bond types and chemical groups known to thoseskilled in the art. The linker may consist of the amino acid (s)comprising X⁻¹. The linker may remain on the therapeutic drug, or may beremoved soon thereafter, either by further reactions or in aself-cleaving step. Self-cleaving linkers are those linkers which canintramolecularly cyclize and release the drug or undergo spontaneousS_(N)1 solvolysis and release the drug upon peptide cleavage.

Other materials such as detectable labels or imaging compounds can belinked to the peptide. Groups can be linked to the amino terminus of X₇,including such moieties as antibodies, and peptide toxins, including the26 amino acid toxin, melittin and the 35 amino acid toxin cecropin B forexample. Both of these peptide toxins have shown toxicity against cancercell lines. The N-terminal amino acid of the peptide may also beattached to the C-terminal amino acid either via an amide bond formed bythe N-terminal amine and the C-terminal carboxyl, or via coupling ofside chains on the N-terminal and C-terminal amino acids or viadisulfide bond formed when the N-terminal and C-terminal amino acidsboth consist of the amino acid cysteine. Further, it is envisioned thatthe peptides described herein can be coupled, via the carboxy terminusof X₁ or X⁻¹, to a variety of peptide toxins (for example, melittin andcecropin are examples of insect toxins), so that cleavage by hK2liberates an active toxin. Additionally, the peptide could be coupled toa protein such that the protein is connected at the X₁ or X⁻¹ amino acidof the peptide. This coupling can be used to create an inactiveproenzyme so that cleavage by a tissue-specific protease (such as hK2 orPSA) would cause a conformational change in the protein to activate it.For example, Pseudomonas toxin has a leader peptide sequence which mustbe cleaved to activate the protein. Additionally, the peptide sequencecould be used to couple a drug to an antibody. The antibody could becoupled to the N-terminus of the peptide sequence (that is, X₄ or higherX amino acids), and the drug coupled to the carboxy terminus (that is X₁or X⁻¹). The antibody would bind to a cell surface protein andtissue-specific protease present in the extracellular fluid could cleavethe drug from the peptide linker

The preferred amino acid sequence can be constructed to be highlyspecific for cleavage by hK2. In addition the peptide sequence can beconstructed to be highly selective towards cleavage by hK2 as comparedto purified extracellular and intracellular proteases. Highly-specifichK2 sequences can also be constructed that are also stable towardcleavage in human sera.

The peptides of the invention can be synthesized according to any of therecognized procedures in the art, including such commonly used methodsas t-boc or fmoc protection of alpha-amino groups. Both methods involvestepwise syntheses whereby a single amino acid is added at each stepstarting from the C-terminus of the peptide. Peptides of the inventioncan also be synthesized by well-known solid phase peptide synthesismethods. Peptides can be characterized using standard techniques such asamino acid analysis, thin layer chromatography, or high performanceliquid chromatography, for example.

The invention encompasses isolated nucleic acid molecules encoding thehK2-specific peptides of the invention, vectors containing these nucleicacid molecules, cells harboring recombinant DNA encoding thehK2-specific peptides of the invention, and fusion proteins that includethe hK2 specific peptides of the invention. Especially preferred arenucleic acid molecules encoding the polypeptides described herein.

Prodrug Compositions

The invention also features prodrug compositions that consist of atherapeutic drug linked to a peptide containing a cleavage site that isspecific for hK2 or any enzyme that has the enzymatic activity of hK2.As noted above, the peptides of the invention can be used to targettherapeutic drugs for activation within hK2 producing tissue. Thepeptides that are useful in the prodrugs of the invention are thosedescribed above.

The therapeutic drugs that may be used in the prodrugs of the inventioninclude any drug which can be directly or indirectly linked to thehK2-specifically cleavable peptides of the invention. Preferred drugsare those containing a primary amine. The presence of the primary amineallows for formation of an amide bond between the drug and the peptideand this bond serves as the cleavage site for hK2. The primary aminesmay be found in the drugs as commonly provided, or they may be added tothe drugs by chemical synthesis.

Certain therapeutic drugs contain primary amines and are among thepreferred agents. These include the anthracycline family of drugs, thevinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides,the pteridine family of drugs, diynenes, the podophyllotoxins, and thetaxanes. Particularly useful members of these classes include, forexample, doxorubicin, daunorubicin, carminomycin, idarubicin,epirubicin, aminopterin, methotrexate, methopterin, mitomycin Cporfiromycin, 5-flurouracil, cytosine arabinoside, etoposide, melphalan,vincristine, vinblastine, vindesine, 6-mercaptopurine, and the like.

Other therapeutic drugs are required to have primary amines introducedby chemical or biochemical synthesis, for example sesquiterpene-lactonessuch as thapsigargin, and thapsigargin and many others know to thoseskilled in the art. The thapsigargins are a group of natural productsisolated from species of the umbelliferous genus Thapsia. The termthapsigargins has been defined by Christensen, et al., Prog. Chem. Nat.Prod., 71(1997) 130-165. These derivatives contain a means of linkingthe therapeutic drug to carrier moieties, including peptides andantibodies. The peptides and antibodies can include those whichspecifically interact with antigens including hK2. The interactions caninvolve cleavage of the peptide to release the therapeutic analogs ofsesquiterpene-γ-lactones. Particular therapeutic analogs orsesquiterpene-γ-lactones, such as thapsigargins, are disclosed in U.S.patent application Ser. No. 09/588,822, filed Jun. 7, 2000, entitled“Tissue Specific Prodrug,” and U.S. patent application Ser. No.09/588,921, filed Jun. 7, 2000, entitled “Tissue Specific Prodrug,” bothof which are incorporated herein in their entireties.

For example, thapsigargins with alkanoyl, alkenoyl, and arenoyl groupsat carbon 8 or carbon 2, can be employed in the practice of theinvention disclosed herein. Groups such asCO—CH═CH)_(n1)—(CH₂)_(n2)—Ar—NH₂, CO—(CH₂)_(n2)—(CH═CH)_(n1)—Ar—NH₂,CO—(CH₂)_(n2)—(CH═CH)_(n1)—CO—NH—Ar—NH₂ andCO—(CH═CH)_(n1)—(CH₂)_(n2)—CO—NH—Ar—NH₂ and substituted variationsthereof can be used as carbon 8 substituents, where n1 and n2 are from 0to 5, and Ar is any substituted or unsubstituted aryl group.Substituents which may be present on Ar include short and medium chainalkyl, alkanoxy, aryl, aryloxy, and alkenoxy groups, nitro, halo, andprimary secondary or tertiary amino groups, as well as such groupsconnected to Ar by ester amide linkages. In other embodiments ofthapsigargin analogs, these substituents groups are represented byunsubstituted, or alkyl-, aryl-, halo-, alkoxy-, alkenyl-, amido-, oramino-substituted CO—(CH₂)_(n3)—NH₂, where n3 is from 0 to 15,preferably 3-15, and also preferably 6-12. Particularly preferredsubstituent groups within this class are 6-aminohexanoyl,7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl, 10-aminodecanoyl,11-aminoundecanoyl, and 12-aminododecanoyl. These substituents aregenerally synthesized from the corresponding amino acids,6-aminohexanoic acid, and so forth. The amino acids are N-terminalprotected by standard methods, for example Boc protection.Dicyclohexylcarbodiimide (DCCI)-promoted coupling of the N-terminalprotected substituent to thapsigargin, followed by standard deprotectionreactions produces primary amine-containing thapsigargin analogs.

The substituents can also carry primary amines in the form of an aminoamide group attached to the alkanoyl-, alkenoyl-, or arenoylsubstituents. For example, C-terminal protection of a first amino acidsuch as 6-aminohexanoic acid and the like, by standard C-terminalprotection techniques such as methyl ester formation by treatment withmethanol and thionyl chloride, can be followed by coupling theN-terminal of the first amino acid with an N-protected second amino acidof any type.

The peptide and therapeutic drug are linked directly or indirectly (by alinker) through the carboxy terminus of the amino acid at X₁ or X⁻¹. Thesite of attachment on the therapeutic drug must be such that, whencoupled to the peptide, the non-specific toxicity of the drug issubstantially inhibited. Thus the prodrugs should not be significantlytoxic.

The prodrugs of the invention may also comprise groups which providesolubility to the prodrug as a whole in the solvent in which the prodrugis to be used. Most often the solvent is water. This feature of theinvention is important in the event that neither the peptide nor thetherapeutic drug is soluble enough to provide overall solubility to theprodrug. These groups include polysaccharides or other polyhydroxylatedmoieties. For example, dextran, cyclodextrin, starch and derivatives ofsuch groups may be included in the prodrug of the invention.

Thapsigargin Analogs

The invention also features derivatized thapsigargin analogs with thederivatization including providing the molecule with a residuesubstituted with a primary amine. The primary amine can be used to linkthe derivatized thapsigargin analog with various other moieties. Amongthese are peptides which link to the analog to give prodrugs withoutsignificant non-specific toxicity, but enzymatic reaction with hK2affords the toxic drug. These enzymatic reactions can liberate thenon-specific toxic thapsigargin derivative, for example by cleavagethrough proteolysis or hydrolysis, various reactions of the side chainsof the eptide, or other reactions which restore the non-specifictoxicity of the thapsigargin analog. These reactions can serve toactivate the derivatized thapsigargin analog locally at hK2 producingtissue, and with relative exclusivity to regions in which theseenzymatic reactions take place. The primary amine containingthapsigargin analog can also be linked to an antibody, oligonucleotide,or polypeptide which binds to an epitope or receptor in the targettissue.

Thapsigargin is a sesquiterpene-γ-lactone having the structure disclosedin International Publication No. WO 98/52966. Primary amines can beplaced in substituent groups pendant from either C-2 or C-8 carbon(carbons are numbered as described in International Publication No. WO98/52966). Preferred primary amine containing thapsigargin analogs thatcan be coupled to the peptides described above include those describedpreviously by the inventors (“Tissue Specific Prodrug” InternationalPatent Application PCT/US98/10285, published as InternationalPublication No. WO 98/52966, corresponding to U.S. Ser. No. 60/047,070and 60/080,046, filed May 19, 1997 and Mar. 30, 1998). These primaryamine-containing analogs have non-specific toxicity toward cells. Thistoxicity is measured as the toxicity needed to kill 50% of clonogeniccells (LC₅₀). The LC₅₀ of the analogs of this invention is desirably atmost 10 μM, preferably at most 2 μM and more preferably at most 200 nMof analog.

Methods of Treatment Using Prodrugs

The invention also provides methods of treatment of treatinghK2-producing cell proliferative disorders of the invention with theprodrugs of the invention. The prodrugs of the invention and/or analogsor derivatives thereof can be administered to any host, including ahuman or non-human animal, in an amount effective to treat a disorder.

The prodrugs of the invention can be administered parenterally byinjection or by gradual infusion over time. The prodrugs can beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, or transdermally. Preferred methods fordelivery of the prodrug include intravenous or subcutaneousadministration. Other methods of administration will be known to thoseskilled in the art.

Method of Producing Prodrugs

The invention provides a method of producing the prodrugs of theinvention. This method involves linking a therapeutically active drug toa peptide of the invention described above. In certain embodiments thepeptide is linked directly to the drug; in other embodiments the peptideis indirectly linked to the drug via a linker. In each case the carboxyterminus of the peptide is used for linking. That is, in an amino acidsequence X₅X₄X₃X₂X₁, the link is established through X₁. If X⁻¹ islinked to the carboxy terminus of X₁, the carboxy terminus of X⁻¹ isused for linking. The therapeutic drug contains a primary amine tofacilitate the formation of an amide bond with the peptide. Manyacceptable methods for coupling carboxyl and amino groups to form amidebonds are know to those skilled in the art.

The peptide may be coupled to the therapeutic drug via a linker.Suitable linkers include any chemical group which contains a primaryamine and include amino acids, primary amine-containing alkyl, alkenylor arenyl groups. The connection between the linker and the therapeuticdrug may be of any type know in the art, preferably covalent bonding.

In certain embodiments, the linker comprises an amino acid or amino acidsequence. The sequence may be of any length, but is preferably between 1and 10 amino acids, most preferably between 1 and 5 amino acids.Preferred amino acids are leucine or an amino acid sequence containingthis amino acid, especially at their amino termini.

Method of Screening Tissue and Determining hK2 Activity

In another aspect the invention provides a method of detectinghK2-producing tissue using peptides of the invention, as describedabove. The method is carried out by contacting a detectably labeledpeptide of the invention with target tissue for a period of timesufficient to allow hK2 to cleave the peptide and release the detectablelabel. The detectable label is then detected. The level of detection iscompared to that of a control sample not contacted with the targettissue. Many varieties of detectable labels are available, includingoptically based labels such as chromophoric, chemiluminescent,fluoresecent or phosphorescent labels and radioactive labels, such asalpha, beta, or gamma emitting labels. In addition a peptide labelconsisting of an amino acid sequence comprising X⁻¹ can be utilized fordetection such that release of the X⁻¹ label by hK2 proteolysis can bedetected by high pressure liquid chromatography. The peptide sequencesof the invention can also be incorporated into the protein sequence of afluorescent protein such that cleavage of the incorporated hK2 specificsequence by hK2 results in either an increased or decreased fluorescentsignal that can be measured using the appropriate fluorometric measuringinstrument.

The invention provides a method for detecting a cell proliferativedisorder that comprises contacting an hK2-specific peptide with a cellsuspected of producing hK2. The hK2 reactive peptide is labeled by acompound so that cleavage by hK2 can be detected. For purposes of theinvention, a peptide specific for hK2 may be used to detect the level ofenzymatically active hK2 in biological tissues such as saliva, blood,urine, and tissue culture media. In an embodiment of the method aspecific hK2 inhibitor is used to confirm that the activity beingmeasured is solely due to peptide cleavage by hK2 and not secondary tonon-specific cleavage by other proteases present in the biologicaltissue being assayed. Examples of hK2 inhibitors that can be employed inthe method include the addition of zinc ions, or the addition of hK2specific antibodies that bind to the catalytic site of hK2 therebyinhibiting enzymatic activity of hK2.

Method of Screening Prodrugs

The invention also provides a method of selecting potential prodrugs foruse in the invention. The method generally consists of contactingprodrugs of the invention with hK2-producing tissue and non-hK2producing tissue in a parallel experiment. The prodrugs which exerttoxic effects in the presence of hK2-producing tissue, but not in thepresence of non-hK2 producing tissue are suitable for the uses of theinvention.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Production of Recombinant hK2

Recombinant hK2 was produced and purified as described in Lovgren etal., Biochem. Bioph. Res. Co., 238, 549 555 (1997). Semenogelin 1 and 11were isolated from human semen as described previously in Malm et al,Eur. J. Biochem., 238, 48 53 (1996). The tripeptide aminomethylcoumarin(AMC) substrates Boc-Phe-Ser-Arg-AMC (SEQ ID NO: 53),Boc-Gln-Gly-Arg-AMC (SEQ ID NO: 54), H-Pro-Phe-Arg-AMC (SEQ ID NO: 55),boc-Val-Pro-Arg-AMC (SEQ ID NO: 56), H-D-Val-Leu-Lys-AMC (SEQ ID NO:57), Tos-Gly-Pro-Arg-AMC (SEQ ID NO: 58), Tos-Gly-Pro-Lys-AMC (SEQ IDNO: 59), Z-Leu-Leu-Arg-AMC (SEQ ID NO: 60), Z-Val-Val-Arg-AMC (SEQ IDNO: 61), Z-Ala-Arg-Arg-AMC (SEQ ID NO: 62), and H-Arg-Gln-Arg-Arg-AMC(SEQ ID NO: 63) were from Bachem (Bubendorf, Switzerland). Theheptapeptide substrates Mu-Ala-Pro-Val-Leu-Ile-Leu-Ser-Arg-AMC (SEQ IDNO: 64) and Mu-Val-Pro-Leu-Ile-Gln-Ser-Arg-AMC (SEQ ID NO: 65)corresponding to the pro peptides of PSA hK2 were from Enzyme SystemsProduct (Livermore, Calif., USA). ACT was purified from human bloodplasma as described in Christensson et al., Eur. J. Biochem., 194, 75563 (1990). PCI was provided by Prof. Johan Stenflo (Malmo UniversityHospital, Malmo, Sweden), and SLPI, and PSTI by Prof. Kjell Ohlsson(Malmo University Hospital, Malmo, Sweden). Benzamidine hydrochloridewas from Amresco® (Solon, Ohio, USA), leupeptin and antipain were fromICN Biomedicals (Costa Mesa, Calif., USA), Aprotinin was from Sigma (St.Louis, Mo., USA), and PPACK from Calbiochem (La Jolla, Calif., USA).

Example 2 Determination of hK2 Cleavage Sites in Semenogelin I and II

Purified semenogelin 1 and 11 (40 μg), was incubated with hK2 (8 μg) in50 mM Tris pH 7.5, 0.1 M NaCl, 0.15 M urea at 37° C. for 4 hours. Thefragments generated were purified by reverse phase HPLC using a C-8column. Elution was achieved with a 0-30% (0.25%/min.) linearacetonitrile gradient and fractions corresponding to individual peakswere collected. The amino terminal sequences of the individual peakswere determined by automated amino terminal sequencing with an AppliedBiosystems 470 A gas-phase sequencer. Cleavage of either Sg I or Sg IIwith hK2 results in generation of a multitude of peptides. After partialseparation of the peptides by reversed phase HPLC on a C-8 column weobtained sequences of four cleavage sites in Sg I and seven cleavagesites in Sg II. The semenogelins contain three types of internalrepeats, as described in Lilja et al., J. Biol. Chem., 264, 1894 2000(1989) and Lilja et al., PNAS USA, 89, 4559 63 (1992). Most of theidentified hK2 cleavage sites were located in different positions inthese repeats. The position and sequence of the cleavage sites in Sg Iand Sg II are shown in FIG. 1, where the cleavage sites are alignedunderneath the arrows. Three identical sites of cleavage in repeat typeI, which occurs twice in SgI and four times in SgII, were identified atpositions 274 and 334 in Sg I and position 454 in Sg II. All but one ofthe cleavage sites contained arginine at positon P1, except for one ofthe cleavages in Semenogelin II, which occurred on the carboxy terminalside of a histidine. It is noteworthy that no cleavages occurred on thecarboxy terminal side of a lysine. Five of the eleven cleavage sitesdetermined were double basic, the amino acid at P2 being eitherarginine, lysine or histidine, indicating that hK2 may cleave substratesat both mono- and di-basic sites. In one case P2 was occupied byphenylalanine which is found in the same position in PCI. In additionglycine, valine, serine, glutamine and aspartate were found at P2. Inmost cleavage sites P3 was occupied by a large group; in six of thecleavages it was glutamine or glutamate and in the other serine,histidine or lysine. In one case alanine was found at P3. When lookingat common motifs if can be seen that in seven cases serine was found inP6. Basic amino acids were found in addition to positions P1 and P2 oncein P5, twice in P3, P4, P6 and P8, and four times in P7. On the carboxyterminal side of the cleavage site leucine was found five times in P-1and tyrosine four times in position P3.

Example 3 pH Dependence of the Enzymatic Action of hK2

The pH dependence of hK2 was determined using a universal buffercomposed of 29 mM citric acid, 29 mM KH₂PO₄, 29 mM boric acid, 0.1 MNaCl and 0.2% bovine serum albumin (BSA). The buffering range is pH2.4-11.8. The rate of the cleavage of the substrate 1-1295 (100 μM) by1.6 pmol hK2 was followed for 20 minutes at pH 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, and 10.

All experiments were done close to physiological pH, at pH 7.5, which isvery close to the pH optimum of hK2.

Example 4 Determination of Kinetic Constants

The measurements were done with Fluoroscan II (Labsystems, Helsinki,Finland) using a 355 nm excitation filter and a 460 nm emission filter.The fluorescence of 7-amino-4-methylcoumarin (AMC) (Sigma, St. Louis,Mo., USA) was determined to be 700 FIU/nmol and this value was used inthe calculations of the rate of product formation. Unless otherwiseindicated all final analyses were performed in 200 μl of the buffer 50mM Tris pH 7.5, 0.1 M NaCl, and 0.2% BSA at 37° C. using 1.6 pmol hK2.BSA was added to the reaction mixture in order to minimize adsorption ofthe enzyme to the walls of the microtiter wells. The amount of hK2 wasquantified by a commercial PSA immunoassay (Prostatus, Wallac, Turku,Finland) with the Mabs H1 17 and HSO which recognize PSA and hK2 equallywell (Lovgren et al., Biochem. Biophys. Res. Co., 213, 888-895 (1995)).Under the conditions described, 1.6 pmol of the hK2 preparation cleaved50 pmol/min of the 100 μM substrate Pro-Phe-Arg-AMC (SEQ ID NO: 55).During the 20 minute measurement time the consumption of the substratesis <7% of their total amount and was not considered to affect thereaction rate. Initial analysis of the substrates were performed with3.2 pmol hK2 at a substrate concentration of 250 μM. K_(m) value for thesubstrates cleaved by hK2 at these conditions were determined using atleast four substrate concentrations ranging from 0.2×K_(m) to 5×K_(m).The K_(m) and k_(cat) values were calculated from Lineweaver-Burk plots.

Substrates ending in either arginine or lysine were tested. The kineticconstants for hydrolysis of the substrates by hK2 are shown in Table 1.The best substrate was the kallikrein substrate Pro-Phe-Arg-AMC (SEQ IDNO: 55) having the highest k_(cat) and k_(cat)/K_(m) values. Thecathepsin B substrate Ala-Arg-Arg-AMC (SEQ ID NO: 62) was also cleavedquite effectively having a relatively high k_(cat) value and a low K_(m)resulting in a four times lower k_(cat)/K_(m) value than that obtainedfor the kallidrein substrate Pro-Phe-Arg-AMC (SEQ ID NO: 55). However,no hydrolysis of Arg-Gln-Arg-Arg-AMC (SEQ ID NO: 63) was detected. HK2cleaved additionally Val-Pro-Arg-AMC (SEQ ID NO: 56), andLeu-Leu-Arg-AMC (SEQ ID NO: 60), but with lower efficiency. As with thesemenogelins, hK2 also here cleaves substrates with Arg at position P1and preferentially a large residue or another Arg at position P2. Noneof the substrates with lysine in the C-terminal position were cleaved.

TABLE 1 Substrate Hydrolysis by hK2 Substrates K_(m) (μM)K_(cat) (min⁻¹) K_(cat)/k_(m) (μM⁻¹min⁻¹) Activity (%) Pro Phe Arg-AMC40 55 1.375 100 (SEQ ID NO: 55) Val Pro Arg-AMC 48 1.6 0.034 6(SEQ ID NO: 56) Gly Pro Arg-AMC NR (SEQ ID NO: 58) Gly Pro Lys-AMC NR(SEQ ID NO: 59) Leu Leu Arg-AMC 71 2.4 0.034 7 (SEQ ID NO: 60)Val Val Arg-AMC NR (SEQ ID NO: 61) Val Leu Lys-AMC NR (SEQ ID NO: 57)Phe Ser Arg-AMC NR (SEQ ID NO: 53) Gln Gly Arg-AMC NR (SEQ ID NO: 54)Ala Arg Arg-AMC 20 7.2 0.360 33 (SEQ ID NO: 62) Arg Gln Arg Arg-AMC NR(SEQ ID NO: 63)

The activity listed in Table 1 is hydrolytic activity of hK2 with 100 μMsubstrate in relation to the hydrolytic activity of hK2 with 100 μM ofthe tissue kallikrein substrate H-Pro-Phe-Arg-AMC (SEQ ID NO: 55). Theentry “NR” means that no reaction was detected.

Example 5 Inhibition of hK2

Activity of hK2 (1.6 pmol) was monitored using the substrateH-Pro-Phe-Arg-AMC (SEQ ID NO: 55) (90 μM). Inhibitors, at commonly usedconcentrations, and hK2 (8.3 nM) were mixed and proteolysis of 90 μMH-Pro-Phe-Arg-AMC (SEQ ID NO: 55) was followed up to 20 minutes,starting directly or 10 minutes after mixing the enzyme with variousinhibitors. Inhibition was evaluated by comparison with enzyme-freecontrols.

The effects of several protease inhibitors on the hydrolytic activity ofhK2 are shown in Table 2. The proteolytic activity is expressed inpercentage of inhibitor-free control after 10 minutes of incubation.

TABLE 2 Effects of Protease Inhibitors on hK2 Activity Inhibitor FinalConcentration (μM) Activity (%) ZnCl₂ 200 1.2 100 10 PCl 0.08 0 0.016 50PPACK 5 0 Benzamidine 20,000 8 5000 27 Leupeptin 100 12 Antipain 500 10100 33 Aprotinin 5 10 30 33 SLPI 4 35 0.08 92 PSTI 4 89 0.08 100 ACT 0.8100

None of the reversible protease inhibitors fully inhibited 8 nM hK2 when90 μM substrate was used. HK2 was only weakly inhibited by thereversible peptide inhibitors leupeptin and antipain. The highestrecommended working concentration (100 μM) of the respective inhibitorwas found to give approximately 60% and 90% inhibition of hK2 activityagainst the 90 μM peptide substrate. Aprotinin proved not to be good hK2inhibitor and benzamidine is required at concentrations above 20 mM forefficient inhibition. The irreversible thrombin inhibitor PPACKinhibited hK2 rapidly when used at a 5 μM concentration. Therefore,PPACK can be used to obtain fast irreversible inhibition of hK2. ZnCl₂effectively inhibits hK2 but when used at high concentrations, easilycauses precipitation of proteins. Of the protease inhibitors present inthe prostate, PSTI and SLPI inhibited hK2 weakly and this inhibition isprobably not physiologically significant. This reaction is however slowand no inhibition of the hK2 activity by a 100-fold molar excess of ACTwas detected during the 20 minute measurement time.

Example 6 Kinetic Analysis of hK2 Inhibition by Zinc

The inhibition of hK2 by Zn²⁺ was studied using the substratesPro-Phe-Arg-AMC (SEQ ID NO: 55) and Ala-Arg-Arg-AMC (SEQ ID NO: 62) atconcentrations varying from 9 to 180 μM and ZnCl₂ concentrations rangingfrom 0.5 μM to 1 mM. Since BSA contains several binding sites for Zn²⁺,it could not be used in the kinetics buffer during zinc inhibitionexperiments. The binding of hK2 to the microtiter well walls caused aconstant decrease in the reaction rate, which was however similar to allzinc concentrations. The velocities were calculated from a five-minutemeasurement time after mixing of the enzymes with the buffer containingsubstrate and zinc.

The enzymatic activity of hK2 was inhibited by zinc ions at micromolarconcentrations, and the inhibition was totally reversed by addition ofEDTA. The inhibition of hK2 by zinc was first tested againstcompetitive, uncompetitive, mixed, non-competitive, and partialnon-competitive inhibitor models using commonly used formulas describedfor the respective inhibition models. Zn²⁺ both increased the K_(m) anddecreased the V_(max). The Dixon plots ([Zn²⁺]/v) for the inhibitionwere not linear. However, at low zinc ion concentrations the inhibitionpattern looked competitive. The inhibition mechanism is clearly morecomplex than the ones described by the formulas used. Further analysisof the inhibition mechanism was done by deriving the rate equations forvarious more complex mechanisms and analyzing the data by least-squaresbest-fit systems. The possible mechanism required two bound zinc ions,and is presented by Scheme 1. In the best mechanism, the first boundzinc ion does not cause inhibition (k=k′, or k′ was even slightly higherthan k).

For this mechanism, the rate of product formation (v) will be given bythe following equation:

$v = {\frac{S}{Ks}\left( {k + {k^{\prime}\frac{Zn}{{Ki}\; 1}}} \right)e}$

where the concentration of free enzyme e is the total enzymeconcentration divided by the sum of a) {(the zinc concentration dividedby K_(i1)) times (the zinc concentration divided by K_(i2))} and b){(the zinc concentration divided by K_(i1)) plus 1} times {(thesubstrate concentration divided by K_(s)) plus 1}.

The equation fitted the experimental data quite satisfactorily. Theconstant values were K_(i1)=4.6+/−3.9 μM, K_(i2)=3.2+/−0.7 μM, and k=k′.Best fit analyses were accomplished also for mechanisms involvinginactivating dimerizations of the hK2 molecules as zinc ions have beenshown to inhibit the mouse gamma-NGF and to be critical in theassociation of the mouse 7S NGF complex (Pattison et al., Biochemistry,14, 2733 39 (1975)). These mechanisms did not result in a more optimalfit than those in Scheme 2.

Example 7 Kinetic Analysis of hK2 Inhibition by PCI

The progress of the reaction of hK2 (8 nM final concentration) with thesubstrate Pro-Phe-Arg-AMC (SEQ ID NO: 55) was monitored at two differentsubstrate concentrations without or with different concentrations of PCI(80, 40 or 16 nM final concentration). The fluorescence measurementswere started directly after mixing the enzyme with the inhibitor. Theinhibitor of hK2 by PCI could be described by the slow-bindinginhibition mechanism presented in Scheme 2, which has been used inanalyzing the interaction of PCI with various serine proteases (Hermanset al., Biochem. J, 295, 239 245 (1993), and Hermans et al.,Biochemistry, 33, 5440 44 (1994)). This mechanism assumes that areversible complex is formed between the proteinase and seine proteinaseinhibitor (serpin). The issues justifying the use of the slow bindinginhibition mechanism despite the commonly held view that theseprin-proteinase complex is irreversible has been discussed in moredetail by Hermans et al. (1993)

where E, S, P, and I represent the enzyme, substrate (peptidyl AMC),product (AMC), product (AMC) and inhibitor (PCI) respectively; K_(m) andk_(cat) are Michaelis and catalytic constants for the enzyme substrateinteraction, and K_(i) is the inhibition constant which is equal toL_(diss)/K_(ass), K_(ass) and K_(diss) are the association anddissociation rate constants for the enzyme inhibitor complex. For thismechanism, the progress curve of product formation is given by:

$P = {{v_{s}t} + {\frac{v_{0} - v_{s}}{k^{\prime}}\left( {1 - ^{- {kt}}} \right)}}$

where P is the amount of product at time t, k′ is an apparent firstorder rate constant, and v_(o) and v_(s) are the initial andsteady-state velocities respectively. For the mechanism shown in Scheme2, v_(o) will be independent of the inhibitor concentration, and v_(s)and k′ will vary with the inhibitor concentration according to thefollowing equations:

$V_{s} = \frac{v_{0}}{1 + {I/K_{i}^{\prime}}}$k^(′) = k_(diss) + k_(ass)^(′) ⋅ I = k_(ass)^(′)(K_(i)^(′) + I)

where K′_(i) and k′_(ass) are apparent constants that are related to thetrue constants by the expressions:

$K_{i} = \frac{K_{i}^{\prime}}{\left( {1 + {S/K_{m}}} \right)}$k_(ass) = k_(ass)^(′)(1 + S/K_(m))

The effect of heparin of the association rate of hK2 and PCI was studiedusing 40 nM PCI, 8 nM hK2, and heparin concentrations ranging from 10⁻⁴to 10⁻⁷ M. The effect of the heparin on hK2 activity was analysed bydetermining K_(m) and k_(cat) for the substrate at different heparinconcentrations. Heparin slightly increased the K_(m) of the substrate(data not shown). The increase had no significant effect on thecalculation of the constants.

Example 8 Hydrolysis of hK2 Substrates

Hydrolyses of particular hK2 substrates were carried out at a hK2concentrations of 1 μg/ml in 50 mM Tris buffer, with 0.1 M NaCl, at pH7.8. Serum hydrolysis measurements were carried out in 50% fresh humanserum in 50 mM Tris buffer, with 0.1 M NaCl, at pH 7.8. The singleletter amino acid code was used to designate the peptide sequences usedfor Table 4. The units FU are arbitrary fluorescence units. The entries“U.D.” were for measurements of less than 0.01 FU/hour.

TABLE 4 Hydrolysis of hK2 Substrates Serum Peptide SequencehK2 Hydrolysis Hydrolysis Rate P7 P6 P5 P4 P3 P2 P1 P′1 Rate (FU/hr/m)(FU/hr) G H E Q K R R L (SEQ ID NO: 66) 5966.31 0.17 G G G K A R R L(SEQ ID NO: 67) 4784.22 0.03 G G G K A H R L (SEQ ID NO: 68) 4100.940.09 G P A H Q R R L (SEQ ID NO: 69) 4017.81 0.10 G S K G H F R L(SEQ ID NO: 70) 3029.27 0.04 G S K G H R R L (SEQ ID NO: 71) 2649.96 UDG K D V S R R L (SEQ ID NO: 72) 2316.12 0.08 G S Q N Q R R L(SEQ ID NO: 73) 2100.48 0.05 G S Y P S R R L (SEQ ID NO: 74) 2060.210.09 G S Y P S S R L (SEQ ID NO: 75) 1456.18 0.06 G H E Q K G R L(SEQ ID NO: 76) 650.80 0.04 G S N T E R R L (SEQ ID NO: 77) 592.34 UD GS Y E E R R L (SEQ ID NO: 78) 324.75 0.04 G K D V S G R L(SEQ ID NO: 79) 242.91 0.05 G S N T E K R L (SEQ ID NO: 80) 255.90 0.13G S K G H F H L (SEQ ID NO: 81) 171.47 0.10 G S Q N Q V R L(SEQ ID NO: 82) 193.55 0.03 G P L I L S R L (SEQ ID NO: 83) 118.21 0.07G S Y E E R H L (SEQ ID NO: 84) 42.87 0.09 G K D V S G H L(SEQ ID NO: 85) 67.55 0.05 G G G K A H H L (SEQ ID NO: 86) 70.15 0.05 GS N T E K H L (SEQ ID NO: 87) 80.54 0.03 G P A H Q D R L (SEQ ID NO: 88)75.34 0.06 G H E Q K G H L (SEQ ID NO: 89) 1.30 UD G P A H Q D H L(SEQ ID NO: 90) 48.06 0.00 G S Y P S S H L (SEQ ID NO: 91) 24.68 UD G SQ N Q V H L (SEQ ID NO: 92) 32.48 0.03

TABLE 5 Additional hK2 Substrates Serum Peptide Sequence hK2 HydrolysisHydrolysis Rate P7 P6 P5 P4 P3 P2 P1 P′1 Rate (FU/hr/m) (FU/hr) G H A QK R R L (SEQ ID NO: 93) 3665.1 0.08 G G K S R R L (SEQ ID NO: 94) 3439.70.03 G H E Q K R R L (SEQ ID NO: 66) 3366.5 UD G H E A K R R L(SEQ ID NO: 95) 3324.1 UD G G Q K R R L (SEQ ID NO: 96) 3267.4 0.02 G HE Q K R R A (SEQ ID NO: 97) 3051.5 0.06 G G A K R R L (SEQ ID NO: 98)2773.0 0.02 G H E Q K R R S (SEQ ID NO: 99) 2638.5 UD G G K K R R L(SEQ ID NO: 100) 2583.0 UD G G H K R R L (SEQ ID NO: 101) 2428.4 UD G GK A F R L (SEQ ID NO: 102) 2374.2 0.07 G A E Q K R R L (SEQ ID NO: 103)2325.8 0.10 G G K A Q R L (SEQ ID NO: 104) 2233.7 0.04 G G K A R R L(SEQ ID NO: 105) 2171.2 UD G G K Q R R L (SEQ ID NO: 106) 2171.2 0.02 GG K H R R L (SEQ ID NO: 107) 2079.2 UD G H E Q A R R L (SEQ ID NO: 108)1956.4 0.14 G G K A K R L (SEQ ID NO: 109) 1788.9 0.14 G H E Q K R R dL(SEQ ID NO: 110) 1690.9 0.15 G G K A R R S (SEQ ID NO: 111) 1609.6 UD GG K A R K L (SEQ ID NO: 112) 1602.4 UD G H E Q K R R E (SEQ ID NO: 113)1473.8 UD G G K A H R L (SEQ ID NO: 114) 1287.4 0.10 G G K A N R L(SEQ ID NO: 115) 1113.9 0.01 G G K A R Q L (SEQ ID NO: 116) 1021.9 0.13G G K A R H L (SEQ ID NO: 117) 939.3 UD G G K A R N L (SEQ ID NO: 118)828.4 0.25 G G K A dR R L (SEQ ID NO: 119) 494.4 0.06 G G K A K K L(SEQ ID NO: 120) 77.9 UD G G K A H K L (SEQ ID NO: 121) 73.2 UD G G K AR dR L (SEQ ID NO: 122) 49.6 UD G G K A dR dR L (SEQ ID NO: 123) 16.5 UD

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the forgoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-62. (canceled)
 63. A composition comprising a prodrug, the prodrugcomprising a therapeutically active drug; and a peptide comprising anamino acid sequence having a cleavage site specific for an enzyme havinga proteolytic activity of human kallikrein 2 (hK2), wherein the peptideis linked to the therapeutically active drug to inhibit the therapeuticactivity of the drug, and wherein the therapeutically active drug iscleaved from the peptide upon proteolysis by an enzyme having aproteolytic activity of human kallikrein 2 (hK2), and wherein thepeptide comprises an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 93, SEQ IDNO: 102, and SEQ ID NO:
 104. 64. The composition of claim 63, whereinthe peptide is linked directly to the therapeutic drug.
 65. Thecomposition of claim 64, wherein the peptide is linked directly to aprimary amine group on the therapeutic drug.
 66. The composition ofclaim 63, wherein the peptide is linked to the therapeutic drug via alinker.
 67. The composition of claim 66, wherein the therapeutic drug isa compound belonging to the group of thapsigargins which have beenderivatized with a moiety containing a primary amine group, and thelinker is selected from the group consisting of (a)CO—(CH═CH)_(n1)—(CH₂)_(n2)—Ar—NH₂, (b)CO—(CH₂)_(n2)—(CH═CH)_(n1)—Ar—NH₂, (c)CO—(CH₂)_(n2)—(CH═CH)_(n1)—CO—NH—Ar—NH₂, (d)CO—(CH═CH)_(n1)—(CH₂)_(n2)—CO—NH—Ar—NH₂, (e) CO—(CH₂)_(n3)—NH₂, and (f)CO—(CH₂)_(n3)—NH—CO—CH(R₄)—NH₂, each of which is unsubstituted oralkyl-, aryl-, halo-, alko-, alkenyl-, amido- or amino-substituted, andwherein n1 and n2 are from 0 to 5, n3 is from 0 to 15, Ar is anysubstituted or unsubstituted aryl group, attachment of NH₂ to Ar is in aortho, meta or para position with respect to the remainder of thelinker, and R₄ is any naturally occurring amino acid side chain.
 68. Thecomposition of claim 66, wherein the linker is an amino acid sequence.69. The composition of claim 68, wherein the linker comprises a leucineresidue.
 70. The composition of claim 63, wherein the therapeuticallyactive drug inhibits a sarcoplasmic reticulum and endoplasmic reticulumCa²⁺-ATPase (SERCA) pump.
 71. The composition of claim 70, wherein thetherapeutically active drug is selected from the group of primary aminecontaining thapsigargins and thapsigargin derivatives.
 72. Thecomposition of claim 63, wherein the therapeutically active drugintercalates into a polynucleotide.
 73. The composition of claim 72,wherein the therapeutically active drug is an anthracycline antibiotic.74. The composition of claim 73, wherein the therapeutically active drugis selected from the group consisting of doxorubicin, daunorubicin,epirubicin and idarubicin.
 75. The composition of claim 63, wherein thetherapeutically active drug has an LC₅₀ toward ER Ca²⁺-ATPase of at most500 nM.
 76. The composition of claim 75, wherein the therapeuticallyactive drug has an L₅₀ toward ER Ca²⁺-ATPase of at most 50 nM.
 77. Thecomposition of claim 63, wherein the therapeutically active drug has anLC₅₀ toward hK2-producing tissue of at most 2.0 μM.
 78. The compositionof claim 77, wherein the therapeutically active drug has an LC₅₀ towardhK2-producing tissue of less than or equal to 2.0 μM.
 79. Thecomposition of claim 63, further comprising an added substituent whichrenders the composition water soluble.
 80. The composition of claim 79,wherein the added substituent is a polysaccharide.
 81. The compositionof claim 80, wherein the polysaccharide is selected from the groupconsisting of modified or unmodified dextran, cyclodextrin and starch.82. A method of treating a cell proliferative disorder which produceshK2, the method comprising administering the composition of claim 63 ina therapeutically effective amount to a subject having the cellproliferative disorder.
 83. The method of claim 82, wherein the disorderis benign.
 84. The method of claim 82, wherein the disorder ismalignant.
 85. The method of claim 84, wherein the malignant disorder isprostate cancer.
 86. The method of claim 84, wherein the malignantdisorder is breast cancer.